Antibodies are proteins which exhibit binding specificity to a specific antigen. Native antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a .beta.-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the .beta.-sheet structure. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. Of the various human immunoglobulin classes, only human IgG1, IgG2, IgG3 and IgM are known to activate complement; and human IgG1 and IgG3 mediate ADCC more effectively than IgG2 and IgG4
Papain digestion of antibodies produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. The crystal structure of the human IgG Fc region has been determined (Deisenhofer, Biochemistry 20:2361-2370 (1981)). In human IgG molecules, the Fc region is generated by papain cleavage N-terminal to Cys 226. The Fc region is central to the effector functions of antibodies.
The effector functions mediated by the antibody Fc region can be divided into two categories: (1) effector functions that operate after the binding of antibody to an antigen (these functions involve the participation of the complement cascade or Fc receptor (FcR)-bearing cells); and (2) effector functions that operate independently of antigen binding (these functions confer persistence in the circulation and the ability to be transferred across cellular barriers by transcytosis). Ward and Ghetie, Therapeutic Immunology 2:77-94 (1995).
While binding of an antibody to the requisite antigen has a neutralizing effect that might prevent the binding of a foreign antigen to its endogenous target (e.g. receptor or ligand), binding alone may not remove the foreign antigen. To be efficient in removing and/or destructing foreign antigens, an antibody should be endowed with both high affinity binding to its antigen, and efficient effector functions.
Fc Receptor (FcR)
The interaction of antibodies and antibody-antigen complexes with cells of the immune system effects a variety of responses, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) (reviewed in Daeron, Annu. Rev. Immunol. 15:203-234 (1997); Ward and Ghetie, Therapeutic Immunol. 2:77-94 (1995); as well as Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991)).
Several antibody effector functions are mediated by Fc receptors (FcRs), which bind the Fc region of an antibody. FcRs are defined by their specificity for immunoglobulin isotypes; Fc receptors for IgG antibodies are referred to as FcγR, for IgE as Fc.epsilon.R, for IgA as Fc.alpha.R and so on. Three subclasses of FcγR have been identified in humans: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Because each FcγR subclass is encoded by two or three genes, and alternative RNA spicing leads to multiple transcripts, a broad diversity in FcγR isoforms exists. The three genes encoding the FcγRI subclass (FcγRIA, FcγRIB and FcγRIC) are clustered in region 1q21.1 of the long arm of chromosome 1; the genes encoding FcγRII isoforms (FcγRIIA, FcγRIIB and FcγRIIC) and the two genes encoding FcγRII (FcγRIIIA and FcγRIIIB) are all clustered in region 1q22. These different FcR subtypes are expressed on different cell types (reviewed in Ravetch and Bollard, Annu. Rev. Immunol. 19:275-290 (2001). For example, in humans, FcγRIIIB is found only on neutrophils, whereas FcγRIIIA is found on macrophages, monocytes, natural killer (NK) cells, and a subpopulation of T-cells. Notably, FcγRIIIA is the only FcR present on NK cells, one of the cell types implicated in ADCC.
FcγRI, FcγRII and FcγRII are immunoglobulin superfamily (IgSF) receptors; FcγRI has three IgSF domains in its extracellular domain, while FcγRII and FcγRIII have only two IgSF domains in their extracellular domains.
Another type of Fc receptor is the neonatal Fc receptor (FcRn). FcRn is structurally similar to major histocompatibility complex (MHC) and consists of an .alpha.-chain noncovalently bound to .beta.2-microglobulin.
FcγRII (CD32), has several isoforms, IIa, IIIb1, Iib2, IIb3 and IIc, and is the most widely distributed human FcγR type, being expressed on most types of blood leukocytes, dendritic cells and platelets. FcγRII is a low affinity receptor that only binds to aggregated IgG. It is the only FcγR class to be able to bind to IgG2. The FcγRIIa is expressed on a range of cell types, including monocytes, macrophages, neutrophils, eosinophils and basophils, which it can co-activate in combination with other immunogloblulin receptors through its ITAM motifs. FcγRIIa binds IgG antibodies attached to cells and causes lysis of those cells. This process is called antibody-dependant cell mediated cytotoxicity. The FcγRIIb is also widely expressed but bears an immunoreceptor tyrosine-based inhibitory motif (ITIM) which is necessary for its inhibitory effects. FcγRIIb can suppress activation of B cells by invoking negative signaling when it is cross-linked to surface immunoglobulin via Ab-Ag complexes. The activation of macrophages and monocytes via FcγR is suppressed by co-ligation of FcγRIIb. (Armour et al. (2003) “Differential binding to human FcγRIIa and FcγRIIb receptors by human IgG wildtype and mutant antibodies,” Mol. Immunol., 40:585-593). Cells transfected to express both FcγRIIa and FcγRIIb show reduced phagocytosis relative to cells bearing FcγRIIa alone and the cytotoxicity of anti-tumour Ab was enhanced in FcγRIIb-deficient mice. (Hunter et al. (1998) “Inhibition of Fcγ receptor mediated phagocytosis by nonphagocytic Fcγ receptor.” Blood, 91:1762-1768; and Clynes et al. (2000) “Inhibitory Fc receptors modulate in vivo cytotoxicity against tumour targets.” Nat. Med. 6:443-446). Thus, shifting relative binding affinity of FcγR from FcγRIIa to FcγRIIb, for example would suppress activation of B cells and or macrophages and monocytes and would be useful to dampen immune responses in inflammatory disorders such in various autoimmune diseases. Conversely, shifting the relative binding affinity of Fcγ from FcγRIIb to FcγRIIa would lead to an enhancement of ADCC, and would be useful to enhance tumor cell killing, for example in the treatment of cancer.
FcγRIII (CD16) has two isoforms which are able to bind to IgG1 and IgG3. The FcγRIIIa has an intermediate affinity for IgG and is expressed on macrophages, monocytes, NK cells and subsets of T cells. FcγRIIIb is a low-affinity receptor which is selectively expressed on neutrophils. FcγRIIIa, like FCγRIIa, binds to IgG antibodies attached to cells and causes the lysis of those cells by ADCC. FcγRIIIa binds clustered IgG molecules bound to cell surfaces and does not bind to monomeric IgG. Therefore, ADCC occurs only when the target cell is coated with antibody. Engagement of FcγRIIIa by antibody-coated target cells activates NK cells to synthesize and secrete cytokines such as IFN-γ, as well as discharge the contents of their granules, which mediate the cytolytic functions of this cell type.
Altering the effector activity of antibodies by shifting effector function from otherwise inhibitory immune response to inducing ADCC and vice versa is desirable for bettering treatment outcomes in a variety of diseases and conditions. Lazar et al. (2006) Proc. Natl. Acad. Sci. U.S.A., 103:4005, Stavenhagen et al. (2007) Cancer Res. 67:8882, Oganesyan et al. (2008) Mol. Immunol. 45:1872, Veri et al. (2007) Immunology, 121:392, and Shields, et al. (2001) J. Biol. Chem. 276:6591 disclose efforts in this research area.
Presta et al. (U.S. Pat. No. 6,737,056) discloses polypeptides comprising a variant Fc region. The disclosed variations are based on single position mutations. By performing alanine scans, Presta et al. discloses single position Fc region amino acid modification at positions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439; these single position mutations are purported to result in reduced binding to FcγRII. Presta et al. also discloses that systematic alanine scans of the entire Fc region purportedly show that an Fc region amino acid modification at any one of amino acid positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388, 389, 416, 434, 435 or 437 results in reduced binding to FcγRIII. Presta et al. also discloses polypeptides with increased binding to an FcγRII comprising single position amino acid modifications at any one of amino acid positions 255, 256, 258, 267, 268, 272, 276, 280, 283, 285, 286, 290, 301, 305, 307, 309, 312, 315, 320, 322, 326, 330, 331, 337, 340, 378, 398 or 430 of the Fc region identified by alanine scans as well. Presta et al. discloses one single instance where two Fc amino acid positions are mutated at the same time (i.e., modifications S317A and K353A), however Presta et al. does not suggest that these double mutations provide any sysnergistic effect in obtaining a desired binding profile of the modified polypeptide to FcγRII. Presta et al. is completely silent on potential synergism of simultaneous modifications at more than a single amino acid position.
Lazar et al. (U.S. Pat. No. 7,317,091) discloses antibodies comprising an amino acid modification at position 332 in the Fc region purportedly resulting in altered binding to an FcγR. Lazar et al. discloses that individual substitutions in positions 234, 235, 239, 240, 243, 264, 266, 272, 274, 278, 325, 328, 330, and 332 purportedly effect the binding to an FcγR. While Lazar al. purports to disclose synergy of Fc variants when combined with engineered glycoforms, Lazar et al. is silent on potential synergism that may be provided by a selection of simultaneously modified Fc amino acid positions, regardless of additional synergism that may be provided by engineered glycoforms.
Stavenhagen (U.S. Pat. No. 7,632,497) discloses molecules having a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region. These modified molecules purportedly confer an effector function to a molecule, where the parent molecule does not detectably exhibit this effector function.
Current approaches to optimize the Fc region in therapeutic monoclonal antibodies and soluble polypeptides fused to Fc regions have focused on a limited number of single amino acid changes based on alanine screens, site-directed mutagenesis etc. Other approaches in engineering Fc regions have focused on the glycosylation of the Fc region to optimize Fc region function. Still other approaches have focused on Fc modifications that purportedly confer effector function by modifying wild type molecules that lack effector function, but do not purport to increase or otherwise modify existing effector function of a wild type molecule.
There is currently no known Fc protein or polypeptide that is optimized to bind a particular FcγR of interest with very high specificity as compared to other Fcγ receptors. Although the effect of individual Fc region amino acid mutations on the binding with certain Fcγ receptors is well understood, previous studies fail to describe the effect of simultaneous changes to multiple amino acids. Additionally, the prior art does not suggest suitable replacements for substituted Fc region amino acids to obtain optimal binding to the FcγR of interest, including modification of effector function of a wild type molecule that already has detectable effector function.
Hence, there is a need in the art for polypeptides and antibodies that comprise multiple, synergistic amino acid substitutions in the Fc region such that the antibody or fusion protein is optimized for binding to the FcγR isoform of choice.